Thrombopoietin (TPO), initially cloned as a major regulator of platelet production, plays a pivotal role in hematopoietic stem cell (HSC) biology. Kaushansky et al., Nature, 369:568-571 (1994). Virtually all primitive HSC that display repopulating activity express c-Mpl, the receptor of TPO. Solar et al., Blood, 92:4-10 (1998). TPO alone or in combination with other early acting cytokines, such as stem cell factor (SCF), interleukin 3 (IL-3), or Flt-3 ligand enhance proliferation of primative HSC in vitro. Ku et al., Blood, 87:4544-4551 (1996); Sitnicka et al., Blood, 87:4998-5005 (1996). In vivo studies have confirmed these conclusions. Kimura et al., Proc. Natl. Acad. Sci. U.S.A., 95:1195-1200 (1998). The importance of TPO in stem cell self renewal and expansion was also supported by the clinical observation that mutations of the c-Mpl gene caused congenital amegakaryocytic thrombocytopenia, a disease in which all hematopoietic lineages fail during childhood. Ballmaier et al., Blood, 97:139-146 (2001). It has been found that expansion of HSCs in adult bone marrow is 10 to 20 times less robust in tpo−/− mice following bone marrow transplantation. Exogenously added TPO rescued this defect. Fox et al., J. Clin. Invest., 110:389-394 (2002). These reports indicate that TPO is a major non redundant contributor to self renewal and expansion of HSCs.
Autologous stem cell transplantation (ASCT) is increasingly widely used as a means of reconstituting the bone marrow following the administration of potentially curative, myeloablative, high dose chemotherapy. The basis for this technique is to mobilize HSCs from bone marrow to peripheral blood (using G-CSF+/−priming chemotherapy) from which they are harvested by apheresis. These stem cells, which form a minority of the harvested population, are then capable of reconstituting the bone marrow when reinfused following myeloablative therapy. Stem cells obtained from peripheral blood in this technique appear to be similar to cord blood cells and superior to bone marrow cells in their ability to regenerate bone marrow following myeloablative therapy with time to neutrophil and platelet engraftment of less than 10 days. The most common tumor types in which ASCT is used are myeloma, lymphoma (both Hodgkins Disease and Non-Hodgkins Lymphoma) and Acute Myeloid Leukemia. High dose chemotherapy with ASCT may be increasingly used as first line therapy, particularly in myeloma, but it is also used as salvage therapy following the failure of first line chemotherapy. Such subjects often have been heavily pretreated and thus have bone marrow with impaired hematopoietic potential.
Following the re-infusion of these harvested cells into the subject, there is a period of time during which the subject, e.g., a human patient, is at risk of infection (low neutrophils) and bleeding (low platelets). This period of time varies depending on the number of re-infused stem cells, which in turn depends on the ability to stimulate the expansion of stem cells from bone marrow. Further, some subjects also develop bone marrow failure after an initial period of engraftment.
Stem cell transplantation is also used in an allogeneic setting when peripheral blood stem cells are mobilized and harvested from HLA matched donors. Such allogeneic transplants are less frequently employed than ASCT because of the incidence of graft versus host disease but may be used when it is not possible to obtain sufficient stem cells from the patient. However the use of allogeneic stem cells to obtain partial engraftment in the absence of complete myeloablation (the ‘mini transplant’) may also offer some therapeutic benefit due to a graft versus tumor effect. Another possible use, currently in an extremely small number of patients is in the field of gene therapy where normal allogeneic bone marrow cells or autologous cells transduced with a normal copy of a defective gene may be curative for some inherited disease caused by single gene defects. Allogeneic transplants are also under investigation as a therapeutic option for autoimmune diseases.
Despite the potential utility and simplicity of ASCT, there are significant limitations to its widespread use beyond the expected period of pancytopenia, for which intensive subject support is required to allow the re-infused cells to resume levels of hematopoiesis sufficient to maintain peripheral blood counts. A significant proportion (up to 40%) of transplanted subjects requires prolonged platelet transfusions following transplant (primary failure of engraftment). A smaller group (5-10% in autologous but >20% with allogeneic transplant) develop secondary thrombocytopenia despite initial engraftment, sometimes requiring prolonged transfusions. Failure of engraftment or delayed engraftment is associated with increased mortality, increased healthcare costs and decreased subject quality of life.
There thus exists a need to increase HSC production in such subjects. Studies have demonstrated that administration of TPO to patients results in mobilization of peripheral blood progenitor cells. One study demonstrated the mobilization of colony forming cells from multiple lineages and CD34+ cells into the peripheral blood following multiple dose administration of TPO in combination with G-CSF. Another study identified a 6 fold increase in circulating CD34+ cells 3-7 days after administration of a single dose of TPO in cancer patients with otherwise normal hematopoiesis. In this study, a stem cell enriched subfraction (CD34+Thy+Lin−) was increased nearly 9 fold and the committed megakaryocytic subfraction (CD34+CD41+CD14−) was increased nearly 15 fold. This study suggests that TPO is capable of mobilizing both self-renewing HSC and committed daughter cells from bone marrow. Although the availability of recombinant TPO (rhTPO) has shown promise in increasing HSC production, a need exists for an improvement in TPO therapy by way of the mode of drug delivery
There thus exists a need for small molecule mimetic compounds of TPO that retain substantially the full agonist activity of TPO, while at the same time permitting various modes of administration.
There also exists a need for small molecule mimetic compounds of TPO having reduced immunogenicity relative to one or more of rhTPO and rhIL-11 as well as improved pharmacokinetic profile relative to one or more of rhTPO and rhIL-11.